This application is a competitive renewal of a grant that was awarded four years ago to support a comprehensive test of the hypothesis that genetic variation in genes encoding folate metabolizing enzymes was responsible, at least in part, for the occurrence of neural tube defects (NTDs) among newborns. The original application included a large-scale sequencing component as well as innovative strategies for assessing the impact of nonsynonymous substitutions in these enzymes. To successfully implement this project, we assembled a diverse team of experts in molecular genetics, birth defects epidemiology, and pediatrics. Despite being awarded less funding than originally approved, our team accomplished essentially all of the aims. As a result, we have catalogued a diverse set of mutations, common and rare, in nearly the entire folate pathway and developed novel analytical strategies for such data. Specifically, we have uncovered two provocative, complex genetic signatures, one in Hispanics and another in Caucasians, through analyses based on folate pathway modeling. These signatures show compelling statistical evidence (P-values) for distinguishing cases from controls, and may help explain the mechanistic link between the preventive effects of folate and the occurrence of NTDs - a mechanistic link that has been elusive to scientific inquiry. Interestingly, these signatures are different in each ethnicity suggesting that the underlying disease mechanisms are also different: the Hispanic risk profile points to alterations in purine biosynthesis whereas that in Caucasians implicates homocysteine metabolism. These important observations further support the intriguing epidemiological observations that exist, unexplained in the literature. In the current renewal we strive to advance these findings in three ways. First, we propose replication studies that are sufficiently powered to secure the significance of these associations. Second, these complex signatures offer an excellent opportunity to resolve whether the genetic risk for NTDs is driven by the maternal or fetal genotype. Third, we propose mechanistic studies to empirically determine the metabolic outcome from these genetic risk profiles. Because the Hispanic signature is derived from a combination of common variants, we will use cell lines with relevant genotypes as surrogates to represent a range of risk profiles. We propose to quantify folate pathway intermediates via LC/MS and correlate metabolic profile to risk profile. In addition, we will dissect the contribution of individual alleles to the risk signature by reverting single bases vi precise gene editing techniques. Finally, the genetic risk signature in Caucasians, which implicates homocysteine metabolism, resonates with earlier suggestions of an epigenetic dimension to NTDs. Thus, we propose to explore global DNA methylation in the context of NTDs, which could lead to logical extensions at specific genomic loci.